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Previous Article | Next Article 
Journal of Bacteriology, April 1999, p. 2346-2350, Vol. 181, No. 8
Microbiology Unit, Department of
Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
Received 3 December 1998/Accepted 8 February 1999
Soon after asymmetric septation in sporulating Bacillus
subtilis cells, Soon after the asymmetric septation
that occurs early in the sporulation of Bacillus subtilis,
One can readily envisage three possible (nonexclusive) means by which
one sigma factor might replace another. The usurper might have a higher
affinity for E, it might achieve a higher concentration, or it might
supplant the resident sigma factor through the latter's becoming
inactivated. Although there was much interest some years ago in the
exchange of sigma factors in Bacillus (reviewed in reference
28), relatively few experiments on this topic have
been reported recently. To investigate the mechanism by which
E- Overproduction and purification of proteins.
Plasmid pLC2
(21) was used for the overproduction of
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Replacement of Vegetative
A by
Sporulation-Specific F as a Component of the RNA
Polymerase Holoenzyme in Sporulating Bacillus
subtilis
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
References
F is liberated in the prespore
from inhibition by SpoIIAB. To initiate transcription from its cognate
promoters, F must compete with A, the
housekeeping sigma factor in the predivisional cell, for binding to
core RNA polymerase (E). To estimate the relative affinity of E for
A and F, we made separate mixtures of E
with each of the two sigma factors, allowed reconstitution of the
holoenzyme, and measured the concentration of free E remaining in each
mixture. The affinity of E for F was found to be about
25-fold lower than that for A. We used quantitative
Western blotting to estimate the concentrations of E, A,
and F in sporulating cells. The cellular concentrations
of E and A were both about 7.5 µM, and neither changed
significantly during the first 3 h of sporulation. The
concentration of F was extremely low at the beginning of
sporulation, but it rose rapidly to a peak after about 2 h. At its
peak, the concentration of F was some twofold higher
than that of A. This difference in concentration cannot
adequately account for the replacement of A holoenzyme
by F holoenzyme in the prespore, and it seems that some
further mechanism
perhaps the synthesis or activation of an
anti-A factormust be responsible for this replacement.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
References
F is activated in the prespore (7, 29, 37).
However, other sigma factors besides F are present in
the cell early in sporulation, for example, the vegetative
"housekeeping" A factor and the
postexponential-phase-specific H factor (16),
and these, like F, are capable of binding to core RNA
polymerase (here called core RNAP or E). Although A
activity disappears after asymmetric septation (26),
A protein can be detected during sporulation, albeit not
in a form that copurifies with core RNAP in the way that
A from vegetative cells does (37). In this
study, we sought to discover how, notwithstanding the presence of
A, F is incorporated into the holoenzyme
in the prespore. We did not consider how F is released
from inhibition by SpoIIAB (see references 9, 29, and 37).
F replaces E-A in the prespore, we have
determined the relative affinities of A and
F for E and have measured the intracellular
concentrations of E, A and F. We
concluded that the replacement of E-A by
E-F cannot be explained in terms of either a higher
concentration of F or a greater affinity for E, and we
suggest that an anti-A factor may be synthesized or
activated in sporulating cells at about the time of asymmetric septation.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
References
A
protein. It was transformed into the overexpression host
Escherichia coli BL21(DE3) (38). Freshly
transformed cells were grown at 37°C in 2YT containing 100-µg/ml
ampicillin and 0.4% (wt/vol) glucose. At an
A595 of 0.6 to 0.7, the culture was induced with 0.4 mM isopropyl-
-D-thiogalactopyranoside (IPTG),
transferred to 30°C, and incubated for 3 h with shaking. Growth
at 30°C after induction minimizes the formation of inclusion bodies
and ensures that about 60% of the A protein is soluble.
Cells were harvested and stored at 
20°C. Cell pellets were
resuspended in 20 ml of lysis buffer (100 mM Tris-HCl [pH 8.5], 10 mM
EDTA, 5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride
[PMSF]), sonicated briefly, and then passed through a French press.
Lysates were clarified by centrifugation at 16,000 rpm for 90 min. The
supernatant was loaded on a DEAE-Sepharose column, and a linear 0- to
1-M gradient of NaCl in buffer A (20 mM Tris-HCl [pH 8.5], 0.5 mM
DTT, 0.1 mM EDTA) was applied to the column. The fractions containing
A were pooled and loaded onto a Superdex-75 preparative
gel filtration column equilibrated with buffer B (20 mM Tris-HCl [pH
8.0], 2 mM EDTA, 2 mM DTT, 20 mM MgCl2, 100 mM NaCl) and
eluted in the same buffer. Fractions enriched in A were
applied to a Mono-Q (HR5/5) column attached to a Pharmacia fast protein
liquid chromatograph and eluted with a 0.25- to 0.4-M gradient of NaCl
in a buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA, 0.1 mM DTT)
containing 10% glycerol. Eluted fractions were concentrated with
Centricon-10 concentrators (Amicon) and dialyzed against storage buffer
(10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1 mM DTT, 10 mM
MgCl2, 50 mM NaCl, 50% glycerol). Alternatively, A protein was purified from inclusion bodies by
previously published protocols (3, 21).
F purification, E. coli BL21(DE3)
transformants harboring pEAC (30) were grown, induced, and
broken as previously described (5). Cell extracts were
purified by DEAE-Sepharose chromatography and Superdex-75
chromatography as described for A. A third purification
step involved chromatography on a Mono-Q (HR5/5) column with a shallow
gradient of 0.2 to 0.5 M NaCl in 80 ml of TGED (10 mM Tris-HCl [pH
8.5], 0.1 mM EDTA, 0.1 mM DTT, 10% glycerol). F
fractions were concentrated by Centricon-10 concentrators and dialyzed
versus storage buffer.
-mercaptoethanol, 233 mM NaCl, 10% glycerol, 1 mM
PMSF) and passed through a French press. After Polymin P fractionation and ammonium sulfate precipitation (17), the precipitate was resuspended in 8 ml of TGED containing 0.5 M NaCl and loaded onto a
Sephacryl S-300 (Pharmacia) column. RNA polymerase-containing fractions
were pooled, dialyzed overnight into TGED containing 50 mM NaCl, and
subjected to DNA-cellulose affinity chromatography. Elution was
accomplished with 0.7 M NaCl. Fractions were dialyzed versus TGED (pH
7.0) containing 50 mM NaCl and applied to BioRex 70. The core enzyme
was eluted with a 40-ml linear gradient of 0.5 to 1.0 M NaCl in TGED
(pH 7.0) and dialyzed into storage buffer.
Immobilization of A on the sensor chip
surface.
A (0.2 mg/ml) was dialyzed into
phosphate-buffered saline (pH 7.4) containing 1 mM DTT at 4°C.
A was immobilized on the dextran surface of one flow
cell of sensor chip CM5 by the amine coupling method, and HBS buffer
(10 mM HEPES [pH 7.4], 150 mM NaCl, 0.0005% surfactant P20
[Pharmacia]) was the running buffer throughout. All injections
carried out in the immobilization procedure used a flow rate of 10 µl/min. Dialyzed A was diluted 10-fold into coupling
buffer (10 mM sodium acetate, pH 3.8). The sensor chip was activated by
a 40-µl injection of a 1:1 mixture of N-hydroxysuccinimide
and
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (Pharmacia). Immediately after activation, the diluted A was injected in a volume of 40 to 70 µl. This
injection was followed by a 40-µl injection of 1 M ethanolamine-HCl
(pH 8.5) that acted to block any excess activated dextran. Typical
immobilizations resulted in the attachment of 500 to 1,000 resonance
units (RU) of ligand. Binding experiments were always carried out
within 12 h of immobilization.
Measurement of free E after preincubation with A
or F.
A was immobilized to the
sensor chip as described above. This ligand was used to determine the
concentration of free E after preincubation with A or
F. The running buffer contained 10 mM Tris-HCl (pH 7.5),
10 mM MgCl2, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, and 10%
glycerol; E, A, and F were dialyzed into
this buffer. Samples of 100 nM E were incubated with A
at a range of concentrations (0, 12.5, 25, 50, 100, 250, 500, and 750 nM) for 40 min at room temperature to allow reconstitution of the
holoenzyme prior to sample injection (flow rate, 10 µl/min; injection
volume, 40 µl). A 50-µl injection of running buffer containing 0.5 M NaCl was used to regenerate the sensor chip surface after each
injection. The concentration of free E was calculated by comparing the
maximum number of resonance units of the E-A sensorgram
(after subtraction of the blank flow cell signal) obtained in the
absence of A in the preincubation (100% free E) with
the maximum number of resonance units obtained at increasing
concentrations of A. The concentration of
A required to reduce free E by 50% in the preincubation
(K
A) was calculated from a plot
of the log10 A concentration (x
axis) versus the percentage of free E (y axis). KF was obtained by conducting similar
experiments, with the concentration range of F running
from 0 to 6,000 nM.
Induction of sporulation. B. subtilis SG38 was induced to sporulate as described previously (11). Times (hours) after resuspension in starvation medium are called t0, t1, etc. Efficiency of sporulation was confirmed through monitoring of alkaline phosphatase levels in sporulating cell extracts as described previously (8).
Intracellular protein concentrations.
B. subtilis cell
extracts were prepared by incubating 1-ml cell pellets in 250 µl of
50 mM Tris-HCl (pH 7.5), containing 5-mg/ml lysozyme, 10% glycerol,
and 1 mM PMSF for 10 min at 37°C. A 250-µl volume of sodium dodecyl
sulfate (SDS) sample loading buffer (33) was added to each
incubation mixture, and the samples were boiled for 5 min. Assays of
A, F, and core RNAP were made by
immunoblotting with purified antibodies after the proteins from cell
extracts had been separated by SDS-10% (A and core
RNAP) or 15% (F) polyacrylamide gel electrophoresis
(PAGE). Immunoblotting was carried out as described previously
(29) with the following modification: an alkaline
phosphatase-conjugated secondary antibody (Bio-Rad) was used, and blots
were developed by exposure to alkaline phosphatase reagent (Amersham)
for 5 min (1 ml/blot). Each blot included known volumes of standard
solutions of purified proteins, and the quantities of protein in the
unknown samples were determined by use of a Fluoroimager (Molecular
Dynamics). The ImageQuant package was employed to calculate the
quantities of the unknowns and a set of known samples that covered the
range. For core RNAP, both the 
subunit band and the combined
densities of the and ' subunit bands were used in the
quantitation. Average intracellular protein concentration values
(micromolar) were derived by assuming the number of cells per
microliter of a sporulating culture to be 3.2 × 108
and the volume of each cell to be 1.8 × 10
15 liter;
any inaccuracy in this estimation will, of course, affect all protein
concentrations equally. For each protein, average values were derived
from analysis of five or six different blots.
| |
RESULTS AND DISCUSSION |
|---|
Comparison of the relative affinities of A and
F for E.
One possible means by which
F could replace A would be that the
former has a higher affinity for E than the latter. "Direct displacement" of this kind was suggested by Losick and Pero
(28) as a means by which E (the first mother
cell-specific sigma factor) could replace A early in
sporulation. (At the time of that publication [28], F had not been identified.) To compare the affinities of
A and F for E, we used a back-titration
method, in which we measured the free E remaining in solution after
separate mixtures of E and one of the sigma factors had been allowed to
come to equilibrium with the holoenzyme formed in the mixture. In each
such mixture, we determined the concentration of free E by surface
plasmon resonance, using A as the ligand immobilized to
the sensor chip that responds to E. We first constructed a standard
curve, relating the height of the sensorgram in resonance units to the
concentration of E, by passing increasing concentrations of a pure
solution of E over the chip (Fig. 1). We
then preincubated standard concentrations of E with increasing
concentrations of A in solution to allow holoenzyme
reconstitution and applied each mixture to the immobilized
A. Back titration of the E samples with increasing
amounts of A resulted in a steady decrease in the
concentration of free E reported by the sensor chip. Such experiments
allowed us to determine the concentration of A required
in the preincubation to cause a 50% reduction in the concentration of
free E. This concentration is termed KA.
|
A in the preincubation required to
reduce the height of the 50 nM E sensorgram to the height
characteristic of a 25 nM E sensorgram was found to be 50 nM (mean of
three titrations with a range of 44 to 54 nM). These results are shown
in Fig. 2a (filled circles). When the
concentration of E was 100 nM, the concentration of A
required in the preincubation to reduce the height of the sensorgram for free E to a height corresponding to that of 50 nM was 70 nM (range
of three values, 61 to 83 nM; filled circles in Fig. 2b). From these
data, we calculated that the concentration of A in
solution when E was half saturated with A was 20 to 25 nM (mean, 22.5 nM).
|
F needed to back titrate free E was
much greater than the concentration of A needed. The
average concentration of F required to reduce the
concentration of free E from 50 to 25 nM was 540 nM (range of three
values, 524 to 560 nM), and the average concentration of
F required to reduce the concentration of free E from
100 to 50 nM was 657 nM (range of three values, 561 to 754 nM; open
circles in Fig. 2a and b). The concentration of free F
in solution when E is half saturated with F is therefore
515 to 607 nM (mean, 561 nM). Hence, the affinity of E for
A is 25-fold higher than that for F (561 divided by 22.5).
The validity of this comparison of E-sigma affinities relies on the
assumption that the relatively low affinity of F for E
is not due to the sigma's being partially inactivated. SDS-PAGE
analysis of the F samples revealed that no detectable
degradation of F had occurred during the course of the
sporulation experiments. In addition, the F used was
tested for the ability to bind to SpoIIAB. Surface plasmon resonance
experiments demonstrated that, in the presence of ATP, F
had a Kd for immobilized SpoIIAB of 16 nM, a
value almost identical to the 14 nM found previously for a different
F sample (29). Moreover, native-PAGE analysis
indicated that all of the F sample formed a complex with
SpoIIAB in the presence of ATP (results not shown). Furthermore, in
vitro transcription assays showed that on incubation with core RNAP and
substrate DNA, the preparation of F used in this study
yielded amounts of transcript similar to those produced by other
F preparations (results not shown). Taken together,
these results suggest that the F sample used in the
experiments with E was fully active.
The A preparation was also examined by SDS-PAGE and
appeared to be undegraded. Moreover, it was capable of inhibiting
transcription directed by F when E was limiting (results
not shown). Direct measurement by surface plasmon resonance of the
dissociation constant for the interaction between E and
A yielded a Kd of 3 to 4 nM, very
similar to the value reported for the interaction between E and
70 of E. coli (13). The
possibility cannot be absolutely excluded that a fraction of the
preparation was inactive in binding to core RNAP, but any such
inactivation would lead to an underestimate of the difference in the
affinity of E for the two sigma factors.
One possible reason for the high affinity of A may be
that this factor has an extra N-terminal sequence akin to that of
E. coli 70. Although this N-terminal
extension may not itself be directly involved in the binding of E, its
presence in A may help in stabilization of the
E-A complex; for example, the N-terminal extension may
aid in propagating conformational changes between E and
A in the E-A interaction similar to those
apparent between E. coli E and 70 (14,
42). (Such an effect would be additional to the well-known role
of the extension in preventing binding of the sigma factor to the
promoter in the absence of E [6].) We should point out that the comparison of F with A relies on
the assumption that the two proteins utilize the same binding site on
E, so that when F has bound E, the resulting holoenzyme
is unable to interact with A. This assumption seems
likely, since alternative sigma factors have been shown to compete for
E both in vivo and in vitro (4, 10, 18). E binding has been
shown to involve the same region, region 2.1, of several sigma
factorsE. coli 70 and 32
(23, 24, 35), B. subtilis E
(36), and bacteriophage T4 Gp55 (22). Since all
bacterial sigma factors are closely similar at region 2.1 (27), it seems likely that A and
F use the same site to bind to E.
Intracellular concentrations of A, F,
and core RNAP.
We cannot rigorously exclude the possibility that
the affinities of the relevant proteins in the cell are very different
from those in vitro. However, there is no evidence to support such a
suggestion, and we found that E, A, and F
were highly active in transcription assays in buffers similar to those
used for the surface plasmon resonance experiments (results not shown).
If we therefore assume that the results described in the previous
section give a reasonable approximation to the situation in the cell,
they seem to rule out the possibility that the replacement of
E-A in the prespore by E-F is due to
F's having a higher affinity for the core RNAP. An
alternative possibility was that, after asymmetric septation, the
concentration of F in the prespore was much higher than
that of A. Accordingly, we used quantitative
immunoblotting to measure the intracellular concentrations of
A, E, and F during the first 3 h of
sporulation. We found that the A and core RNAP
concentrations were constant, with A being slightly more
concentrated than core RNAP (Fig. 3). A
similar conclusion was reached for A many years ago
(39). (For the above measurements, the core RNAP
concentration was based on scanning of the subunit band. The core
RNAP concentration based on scanning of the and ' bands together
was 10 to 15% lower [results not shown].) As previously reported
(29), F was undetectable in vegetative cells
(t0 samples), but its concentration rose rapidly
at the time of asymmetric septation, reaching a peak value at
t2 which was some twofold higher than that of
A.
|
A plus F) considerably exceeded the
concentration of E. Given that a large fraction of the RNAP is engaged
in elongating RNA and is thus unavailable for binding to sigma, these
values imply that A and F must be in
competition for a limited number of core RNAP molecules. Similarly, a
recent study has suggested that in E. coli, two sigma factors, 70 and S, compete in stationary
phase for a limited supply of E (10). We note that the
concentration of core RNAP estimated here corresponds to about 8,000 molecules/cell, a value similar to that (4,800 to 8,000 molecules/cell)
reported by Bremer and Dennis (1) for E. coli
grown at a comparable doubling time and a little higher than that
reported for E. coli by Ishihama et al. (19).
To what extent is the interpretation of these findings modified by a
possible asymmetry of distribution of sigma factors between the
prespore and the mother cell? Immunofluorescence experiments with
sporulating cells indicate that F shows no obvious
asymmetry in its location in the sporangium; fluorescence was observed
throughout cells that had already formed their asymmetric septum
(25). Recently, similar immunofluorescence experiments have
been used to monitor the location of A during
sporulation. As with F, A was located
throughout the sporangium and apparently at roughly equal
concentrations in the prespore and the mother cell (10a). We
therefore conclude that the concentrations of A and
F in the prespore are unlikely to be very different from
those measured here in the whole cell. In summary, we can say that, at
its peak soon after asymmetric septation, the F
concentration in the prespore may be, at most, a few fold higher than
that of A but is very unlikely to be high enough to
overcome the unfavorable ratio of the affinities of the two sigma
factors for core RNAP.
Mechanism of replacement of EA by
EF.
Given the above results, how can we account for
the replacement of EA by EF in the
prespore? One possibility is that A is inactivated by a
modification that leaves unaffected both its ability to interact with
antibody and its mobility on polyacrylamide gels. A more likely
possibility is that an anti-A factor is synthesized or
activated during sporulation. A cell constituent that interferes with
the function of A and which is metabolically unstable
was described many years ago (34, 40). An
anti-A factor (e.g., a protein inhibitor) could allow
F and G in the prespore and
E and K in the mother cell to replace
A as a component of the holoenzyme during sporulation.
At the time of germination, A could be freed by specific
loss of its anti-sigma factor, and its concentration could be
supplemented by fresh synthesis of A as described by Qi
et al. (32). Since the completion of the experimental work
described here, a specific anti-70 factor, Rsd, has been
reported to be important in sigma factor exchange in E. coli
(20).
A which could be detected by immunoblotting no
matter from which stage of sporulation the enzyme had been isolated.
However, given that we have shown that the concentration of
A is unchanged throughout the first 3 h of
sporulation (Fig. 3), and given the very low dissociation constant for
the interaction between core RNAP and A (see above), it
may be that the method of preparing the holoenzyme used by Fujita and
Sadaie (12) leads to an association of the core with
A that does not occur in the cell. The presence of an
anti-sigma factor attached to A would not necessarily
prevent such an association, since we have evidence that SpoIIAB, the
anti-sigma factor of F, can associate with the
F holoenzyme (27a; see also reference 31).
Our results have some features in common with those of Williams et al.
(41). Those workers found that the sigma factor encoded by
gene 55 of phage T4 had a lower affinity for E than 70
but nonetheless displaced the latter during phage infection. Subsequent
experiments with this system showed that the ability of the gene 55 product to displace 70 was due, in all probability, to
the synthesis of an anti-70 factor by another gene,
asiA, of T4 (31).
| |
ACKNOWLEDGMENTS |
|---|
We thank J. D. Helmann for providing plasmid pLC2; D. A. Harris for much valuable advice and for reading the manuscript; and J. Errington, R. Losick, and I. Lucet for reading the manuscript.
We thank the Biotechnology and Biological Sciences Research Council and the Medical Research Council for financial support and the Wellcome Trust for providing the BIACore facility.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom. Phone: 44 1865 275302. Fax: 44 1865 275297. E-mail: mdy{at}bioch.ox.ac.uk.
Present address: Department of Molecular and Cellular Biology,
Harvard University, Cambridge, MA 02138.
Present address: Sir William Dunn School of Pathology, University
of Oxford, Oxford, United Kingdom.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
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|
|---|
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,
, 
), and core RNAP based on estimation of the 
) during the first 3 h of sporulation. Samples were
collected and assayed as described in Materials and Methods.